Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a “digestion-free” method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications.

重组腺相关病毒（rAAV）是用于基因治疗的主要载体之一。准确的基因组滴度不仅对于临床剂量至关重要，而且还是许多用于AAV产品表征的分析测定的前提。AAV基因组滴度传统上是通过qPCR确定的；然而，尽管付出了巨大的努力，但测定精度仍不是最佳的。最近，液滴数字PCR（ddPCR）成为一种功能强大的替代方案，可提供出色的准确性和精密度。但是，目前ddPCR不如qPCR广泛可用，并且以较低的吞吐量和较高的成本运行。在本文中，我们介绍了一种改进的qPCR方法，该方法具有两个主要的优化方法：（1）使用AAV参考材料代替质粒DNA作为qPCR标准，（2）通过向标准品和样品制备物中添加5％Tween 20来实施“无消化”方法。该新方法已经用不同血清型，纯化状态和衣壳蛋白转基因的AAV进行了广泛的测试，结果发现该方法具有很高的准确性，精确性和鲁棒性。基因治疗领域的研究人员可以轻松采用这种经过显着改进和简化的测定方法，并可以进一步自动化以实现高通量应用。